Transcription Levels of Pseudomonas aeruginosa Exotoxin A Gene and Severity of Symptoms in Patients with Otitis Externa

Abstract

Polymerase chain reaction (PCR)-based detection and transcription of the gene encoding a potent virulence factor, the exotoxin A, were done on 32 isolates of Pseudomonas aeruginosa belonging to 23 genotypes. These isolates were obtained from 22 patients who were admitted to the emergency room in a medical center during a 5-month period with the diagnosis of either unilateral or bilateral otitis externa. Patients showed symptoms that ranged from mild to severe. PCR amplification of a 396-bp fragment of the gene encoding the exotoxin A was done on extracted DNA. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was performed on extracted RNA to detect exotoxin A gene mRNA transcripts. Quantitation of RT-PCR amplicons from P. aeruginosa isolates associated with mild and severe symptoms was determined by end-point titration of c-DNA and scanning of amplicons with the Storm Gel and Blot Imaging System. Data have shown that all of the 32 isolates of P. aeruginosa carry the exotoxin A gene, and all isolates with the exception of two had the exotoxin A transcription demonstrated by the production of a 396-bp amplicon from RT-PCR-amplified RNA. The remaining two isolates amplified fragments that were slightly smaller than the expected size. Additional studies are needed to characterize these two mRNA transcripts. Transcription levels of exotoxin A gene associated with severe symptoms were significantly more elevated than those associated with mild to moderate symptoms. Studies are under way to determine expression of P. aeruginosa exotoxin A by detecting quantitatively levels of the translated exotoxin A protein produced by isolates associated with severe and mild to moderate symptoms.

Host Translational Inhibition by Pseudomonas aeruginosa Exotoxin A Triggers an Immune Response in Caenorhabditis elegans

“…ToxA is expressed in over 90% of clinical P. aeruginosa isolates, high toxA transcription levels are associated with severe infections (Bjorn et al, 1977; Matar et al, 2002), and toxA deficient strains are attenuated in animal virulence assays (Fogle et al, 2002; Miyazaki et al, 1995; Nicas and Iglewski, 1985; Rahme et al, 1995). Additionally, as shown in the accompanying paper, ToxA inhibits protein synthesis in C. elegans IECs during a PA14 infection (Dunbar et al, 2012).…”

Detection of Exo A Gene in Pseudomonas aeruginosa from Human and Dogs Using Polymerase Chain Reaction

“…The result was disagreed with [19] who was showed that, 30/32 isolate of P. aeruginosa from otitis externa in patients were harbored Exo A gene. Frequent recombination of chromosomal genes between different isolates of P. aeruginosa may have led to the genetic diversity of the organism [39].…”

“…Frequent recombination of chromosomal genes between different isolates of P. aeruginosa may have led to the genetic diversity of the organism [39]. The mechanisms of genetic exchange, including transformation, transduction, and conjugation, affect P. aeruginosa to adapt to changing conditions by acquiring new genetic information [40], also the sequences are absent, or rearranged from isolate to isolate [18] or the gene may not be found in some these isolates [19].…”

New Insights into the Effect of Origanum Extracts on the Gene Expression Profiles of Multidrug-resistant Isolates of Pseudomonas aeruginosa Retrieved from Oreochromis niloticus

“…To verify the virulence of P. aeruginosa MDR strains, specific primers targeting the toxA and exoS virulence genes were selected according to Matar, Ramlawi, Hijazi, Khneisser and Abdelnoor (2002) and Strateva (2008), respectively. Furthermore, the antibiotic extrusion machinery in Pseudomonas isolates and its role in antibiotic resistance was identified using two sets of primers targeting mexA and mexB according to Dumas, Van Delden, Perron and Köhler (2006) and Xavier, Picão, Girardello, Fehlberg and Gales (2010).…”